GEN-MKT-18-7897-A
Feb 29, 2016 | Biopharma, Blogs | 0 comments
Traditionally, the pharmacokinetic profile of biotherapeutics such as insulin glargine, adalimumab, trastuzumab and others, used gold standard LBAs to assess dose-response during drug discovery and development. However, LBAs require a specific antibody reagent to be developed for each mAb variant, a process that is often incompatible with the compressed timeframes encountered during the initial stages of drug development. More recently, LC-MS/MS-based methods have come to the forefront as a feasible approach for the quantification of biotherapeutics in biological matrices, with many of these methods relying on proteolytic digestion of the target mAb and quantification of multiple unique signature peptides, which are equivalent to levels of the whole protein. But, to drive the real biological need, we have to quantify the pharmacologically active or free form of the drug to assess safety, efficacy and proper dosing regimen. Here we present a solution to get the best of both technologies: an LBA strategy to capture the active form of the drug; and an LC-MS assay to selectively quantify the free and circulating drug.
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As an analytical strategy, middle-down mass spectrometry (MS) workflows characterize biotherapeutic proteins by analyzing large, digested protein fragments or defined subunits, rather than fully intact proteins (top-down) or digested peptides (bottom-up). A middle-down strategy combines the strengths of top-down and bottom-up approaches by delivering high sequence coverage and structural specificity while maintaining relatively simple sample preparation. In practice, middle-down analysis enables accurate mass measurement, rapid sequence confirmation, and localization of key post-translational modifications (PTMs) on protein subunits that are directly relevant to product quality.
In biopharmaceutical development, sequence variants (SV) are considered an inherent risk of producing complex proteins in living systems. Sequence variants are unintended changes to the amino acid sequence of a biotherapeutic and can be caused by errors in transcription or translation in the host cell, or cell culture and process conditions. Detailed analysis of SVs is important in process and product development to ensure the drug’s safety and efficacy. Even low‑level sequence variants can have significant implications for product quality, safety, and efficacy, making their accurate detection and characterization a critical requirement across development, process optimization, and regulatory submission.
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